Changes in the energy yielding intermediary pathways are implicated in the hypertrophic gene expression program in the heart and can be related to specific inefficiencies in ATP synthesis.1–6 However, a generalized description of the shift in energy providing metabolism as a simple reduction in fatty acid oxidation with increased carbohydrate use, neglects the balance of carbon flux in and out of the citric acid cycle, and neglects changes in the overall dynamics of lipid utilization/storage that produce physiological effects.4–8 Indeed, alternative routes for carbon influx from glucose to the citric acid cycle have been identified and may appear to compensate for reduced fatty acid oxidation in cardiac hypertrophy.5 However, the potential for further, downstream metabolic adaptations as a consequence of rerouting carbon flux remain poorly understood.

We have recently reported that a compensatory route for oxidation of glycolytic end products in the hypertrophied myocardium is activated and counters the reduced oxidation of fatty acids and glycolytic end products.5 This compensatory influx of carbon into the oxidative pathways is apparently attributable to an increase in the cytosolic isoform of malic enzyme in the pressure overloaded heart, with no change in content of the alternative anaplerotic enzymes (see Figure 1).5 Such an increase in pyruvate carboxylation by malic enzyme, produces malate for transfer into mitochondria via the 2-oxoglutarate-malate carrier and thereby supports increased carbon influx directly to the second span of the citric acid cycle, a process termed “anaplerosis.”5,9 Thus, an alternate route for carbohydrate oxidation through increased anaplerosis was unveiled, in part explaining previously reported discrepancies in carbon flux between glycolysis and the oxidation of glycolytic end products in hypertrophied hearts.10,11 However, the anaplerotic route for maintaining carbohydrate oxidation also induces inefficiencies in the oxidative energy metabolism of the hypertrophied myocardium.

The elevated malic enzyme activity in the hypertrophied heart competes with the normal route of pyruvate oxidation via pyruvate dehydrogenase (PDH). The result of redirecting pyruvate metabolism away from PDH is reduced efficiency in the contribution of carbohydrate to oxidative energy metabolism: (1) anaplerotic entry of carbon units from glucose into oxidative metabolism via carboxylation to malate bypasses reactions for NADH generation from PDH and the first span of the citric acid cycle, reducing the energy yield from carbohydrate oxidation; and (2) in carboxylating pyruvate to form malate, the malic enzyme reaction consumes NADPH for triacylglyceride (TAG) formation and may contribute to the recently observed limitations in TAG formation in hypertrophied hearts in the absence of diabetes or obesity.12

We therefore hypothesized that increasing the activation of PDH relative to malic enzyme would ameliorate the potentially maladaptive increase in anaplerotic carbon influx to the citric acid cycle in hypertrophied rat hearts. The influences of pharmacological activation of PDH on the relative contribution of anaplerosis, the mode of carbon influx into the citric acid cycle, and the otherwise reduced TAG pool of the hypertrophied heart were specifically examined. The results enabled investigation of the metabolic mechanisms that are associated with these inefficiencies in energy metabolism that result from shifting carbohydrate oxidation away from PDH to malic enzyme in pressure overload cardiac hypertrophy. Importantly, the findings implicate increased malic enzyme expression and activity in the hypertrophied myocardium as the reason for observed limitations in endogenous lipid storage.5,6

Materials and Methods

Heart Model

Pressure-overload cardiac hypertrophy was induced by constricting the transverse aorta (hemoclip) of 3-week-old male Sprague–Dawley rats that were anesthetized (pentobarbital 65 mg/kg IP) and intubated.5,6 Natural growth of the animal gradually increases the aortic constriction. Rats enter acute end-stage heart failure at 4 to 6 months postbanding. In sham surgical rats (sham), the aorta was isolated without constriction. At 10 weeks postsurgery, banded and sham animals were heparinized (1000 IU) and anesthetized (100 mg/kg pentobarbital), and hearts were excised. The protocol was approved by the Animal Care Policies and Procedures Committee at the University of Illinois at Chicago (Institutional Animal Care and Use Committee accredited). Animals were maintained in accordance with the NIH Guide for the Care and Use of laboratory Animals by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Analysis of 13C spectra provided the fractional contribution from 13C-enriched substrate to mitochondrial ATP production. The fractional enrichment of acetyl-coenzyme A (CoA) (Fc) entering the citric acid cycle and relative contribution of anaplerosis to the citric acid cycle (y) were determined by isotopomer analysis of the glutamate 3- and 4-carbon 13C resonances.14,15 The contribution of anaplerosis is expressed as the ratio of anaplerotic flux to citric acid cycle flux through citrate synthase.14

1H NMR analysis of alanine 13C fractional enrichment in hearts supplied [1,6-13C2]glucose and unlabeled palmitate was performed for determination of oxidation of glycogen, as previously reported.6,13,16 This factors enrichment of all pyruvate from glycolysis and lactate, via reverse flux through lactate dehydrogenase.13 The remaining balance of unenriched substrates contributing to acetyl-CoA formation is then derived from endogenous fatty acids.6 As previously shown, incorporation of [1,6-13C2]glucose into the glycogen pool is minimal, producing a fractional enrichment of only 3.5%.6 Thus, the contribution from glycogen, as opposed to free [1,6-13C2]glucose, to 13C enrichment of acetyl-CoA is accounted for, yet minimal.

Tissue malate was determined by fluorometric analysis of perchloric acid extracts of frozen left ventricle.17 Myocardial triacylglycerols were extracted with chloroform and methanol, and quantified colorimetrically by enzymatic assay for glycerol (Wako Pure Chemical Industries).6,18,19

Statistical Analysis

Data are presented as means±SE. Comparison of more than 2 group means was performed with ANOVA and a Tukey post test for multiple comparisons. Comparison of 2 group means was performed with Student’s t test. Functional measurements over time were compared using the repeated-measures ANOVA. Differences were considered significant at a probability level of <5% (P<0.05).

Results

Cardiac Hypertrophy

As expected, significant cardiac hypertrophy was produced by 10 weeks postbanding of the aorta. Heart mass was larger than shams in all aortic banded rats by 42%, coupled with a 53% increase in mean heart-to-body ratio (mg/g) for aortic banded rats (P<0.05). The degree of hypertrophy was similar in hearts perfused with or without DCA (Sham=2.0±0.07 g; Hyp=2.83±0.05 g, P<0.05; DCA Sham=2.0±0.05 g, DCA Hyp=2.81±0.3 g, P<0.05).

Hearts in all groups displayed stable functional performance, with no change in rate-pressure-product (RPP), as an index of work, over the duration of the protocol (Figure 2). RPP in hypertrophied hearts was reduced by 13% to 26% throughout perfusion as compared to sham hearts, although the lower mean developed pressure in the left ventricle (LVP) of hypertrophied hearts was not quite statistically significant (Table 1).

DCA did not significantly affect RPP in sham and hypertrophied hearts, as previously noted.20 However, hypertrophied hearts displayed depressed rates of pressure development and relaxation, as assessed by +dP/dt and −dP/dt, respectively (Table 1). Despite no affect on RPP, DCA administration did improve contractility and pressure development in hypertrophied hearts, as shown by +dP/dt, −dP/dt, and LVP (Table 1).

Mediation of Anaplerosis in Hypertrophied Hearts

Hypertrophied hearts displayed elevated malic enzyme expression, as shown by results of Western Blot assay in Figure 3. However, expression of pyruvate carboxylase and propionyl CoA carboxylase was similar between sham and hypertrophied hearts (Figure 3A and 3B). Malic enzyme was elevated in hypertrophied hearts with or without DCA treatment, in comparison to corresponding sham groups (Figure 3C).

As in our previous study, and consistent with the increased malic enzyme expression, glucose entry into the oxidative metabolism of the hypertrophied cardiomyocyte was increased via anaplerosis (Figure 4) without an increase in oxidation via PDH (Table 2).5 Although mean values for glucose oxidation and formation of acetyl-CoA were slightly higher in hypertrophied hearts compared to shams, no significant difference was established (Table 2). However, acetyl-CoA formation from palmitate was 10% lower in hypertrophied hearts, which is consistent with increased glucose oxidation.

As expected, DCA increased glucose oxidation through PDH with commensurate reduction in the contribution from palmitate in both hypertrophied and sham hearts. Table 1 displays the values for the fraction of [1,6-13C2]glucose contributing to acetyl-CoA. Hypertrophied hearts oxidized more glucose when PDH was activated. This was also evident from a relatively large reduction in palmitate oxidation, compared to palmitate oxidation with DCA treatment of sham hearts (Table 2). The potentiated response of the hypertrophied hearts is likely attributable to the increased availability of glucose in the cytosol.11,21

Figure 4 displays the relative contribution of anaplerosis to the citric acid cycle in all four experimental groups. Importantly, increasing competition for anaplerotic recruitment of glycolytic pyruvate by PDH activation did attenuate the observed mean activity of anaplerosis in the hypertrophied myocardium (Figure 4). However, this competition did not completely restore anaplerosis to control levels, in the absence of an actual reduction in malic enzyme in the hypertrophied myocardium (Figure 3C).

Effects on Fatty Acid Metabolism

Consistent with attenuation of flux through malic enzyme following PDH activation, and because of the competition for pyruvate, tissue content of malate, the product of malic enzyme was reduced in hypertrophied hearts receiving PDH activation (Table 3). However, the more surprising finding is that increased competition between activated PDH and malic enzyme recovered TAG levels in the hypertrophied myocardium to control levels (Table 3). As shown in a previous report from our laboratory, myocardial TAG content and turnover were both limited in the pressure-overloaded, hypertrophied rat heart.6 In the present study, TAG content in hypertrophied hearts was normalized by PDH activation. Despite reduced fatty acid oxidation in DCA-treated sham hearts, TAG content was not increased.

Effect of Competition for Pyruvate Between PDH and Malic Enzyme on Malate and Triacylglyceride Content

Interestingly, combined data from protocols 1 and 2 indicate that the endogenous, unlabeled long chain fatty acids, from TAG stores contributed to oxidative metabolism in only the control sham hearts (Table 2). In these sham hearts, the balance of acetyl-CoA units formed from unlabeled fuels was attributable to 10% from the unenriched glucose units from glycogen and 6% from TAG. As previously reported, no contribution from TAG to fatty acid oxidation was evident in the hypertrophied hearts, in which glycogen accounted for the remaining balance of unlabeled acetyl-CoA formation.6 Because of PDH activation in sham hearts treated with DCA, the expected increase in carbohydrate oxidation from both exogenous glucose and endogenous glycogen displaced any apparent contribution from TAG to oxidative metabolism. Hypertrophied hearts treated with DCA also showed no apparent contribution of TAG to fatty acid oxidation. Thus, although PDH activation did exert competition for pyruvate against the increased anaplerosis through malic enzyme (Figure 4) and was effective at restoring TAG content (Table 3) in hypertrophied hearts, the protocol did not restore the contribution of TAG to fatty acid oxidation. Indeed, as in DCA-treated sham hearts, the increased carbohydrate oxidation is likely to have precluded oxidation of the endogenous fatty acids in hypertrophied hearts with PDH activation.

Discussion

This study is the first analysis of mechanisms supporting increased anaplerosis as a compensatory pathway for the previously observed reduction in fatty acid oxidation in the hypertrophied heart.5,6 The protocols tested whether the potentially maladaptive increase in anaplerotic flux of pyruvate, through the malic enzyme reaction in the cytosol of the hypertrophied myocardium, can be managed through competition for pyruvate with PDH activation. The persistent increase in malic enzyme expression by hypertrophied hearts treated with DCA and reduced anaplerotic flux suggests the dominance posttranscriptional regulation. As a consequence of competition between enzymes for substrate, anaplerotic activity was reduced, although not normalized in the presence of persistent malic enzyme content (Figures 3 and 4⇑). Reducing anaplerosis also normalized tissue levels of the malic enzyme product, malate, and TAG in hypertrophied hearts. These findings suggest a maladaptive role for elevated activity of the cytosolic isoform of malic enzyme, that limits TAG formation in cardiac hypertrophy (Table 3). Importantly, the metabolic intervention improved contractility (Table 1 and Figure 2).

The activation of anaplerosis in hypertrophied hearts was effectively reduced, but not completely normalized, by increasing the level of competition between PDH and the elevated malic enzyme for pyruvate as a substrate. That anaplerosis was not quite reduced to control levels, is not surprising, because the protocol provided competition between enzymes without normalizing malic enzyme content in the hypertrophied myocardium (Figure 3C). As a consequence of increased pyruvate oxidation via PDH and less pyruvate carboxylation through malic enzyme, tissue levels of the carboxylation product malate were reduced. The resulting reduction in both anaplerosis and malate content provide further evidence that the mechanism of increased anaplerosis in hypertrophied myocardium stems from increased transcript activity of the cytosolic malic enzyme.5 As stated above, malate production through the cytosolic isoform enters the mitochondria as an anaplerotic carbon source via transport through the bidirectional, 2-oxoglutarate-malate carrier on the mitochondrial membrane. The importance of the involvement of this cytosolic malic enzyme, is that the compartmentation of the enzymatic reaction then holds the potential to influence NADPH availability.

A particularly interesting outcome of this study is the restoration of TAG content in the pressure-overloaded, hypertrophied myocardium subjected to PDH activation and reduced anaplerosis. As reported, the pressure-overloaded myocardium displays not only reduced TAG levels but also significantly reduced contributions of the endogenous TAG pool to fatty acid oxidation by the cardiomyocyte.6 The NADPH-dependent malic enzyme is the cytosolic isoform and catalyzes the carboxylation of pyruvate, in the process consuming NADPH to yield NADP+. Unfortunately, the NADPH assay from myocardium does not discriminate between cytosolic and mitochondrial NADPH, with mitochondrial content masking the cytosolic fraction. Thus, as with NADH, the compartmentation of NADPH is not available for analysis. Nevertheless, cytosolic NADPH reduces fatty acyl dihydroxyacetone phosphate for TAG synthesis and may be compromised by increased competition from the NADPH-consuming reaction of cytosolic malic enzyme.

Indeed, the reverse reaction through malic enzyme is generally characterized as lipogenic in adipocytes and can be induced by high fat diet.22 Thus, evidence provided in this present report suggests that increased anaplerosis in hypertrophied hearts reveals an NADPH consuming role for malic enzyme activity (Figure 1). This anaplerotic flux has the potential to then provide competition for NADPH needed to support TAG synthesis. By reducing malic enzyme production of malate, we are also able to demonstrate restoration of the TAG pool in hypertrophied myocardium.

In normal myocardium, several enzymes and pathways are responsible for providing NADPH, which is required not only for TAG generation but also for maintaining cellular redox via reduced glutathione.23–28 Although sources of mitochondrial and cytosolic NADPH remain somewhat controversial, NADPH-consuming reactions in the cytosol would also then play some role in the cellular redox state. According to the findings of Jain and colleagues, our present results would indicate that anaplerotic activity through NADPH-dependent malic enzyme could be maladaptive in limiting cytosolic NADPH for the reduction of glutathione (Figure 1).24,25

As indicated in our previous study, the increased anaplerosis in hypertrophy accounted for an additional 1.0 μmol/min/g dry weight (2.2 μmol/min per gram in hypertrophy versus 1.2 in controls), roughly 10% of citric acid cycle flux.5 Thus, the significant level of flux recruited through malic enzyme is expected to be greater than, or at the very least similar to, that attributed to the actual low flux rate of glucose 6-phosphate dehydrogenase to produce NADPH and within the pentose phosphate shunt in general.27,28 Therefore, our present findings, in comparison to other published reports, point to a potentially important system that balances NADPH production and oxidation via malic enzyme in hypertrophied cardiomyocytes.

Although a mechanism for reduced TAG content in hypertrophied myocardium can be attributed to malic enzyme activity and the link to increased anaplerosis, the severe reduction in the oxidation of fatty acids from the endogenous TAG pool is less clear. A portion of the reduced fatty acid oxidation in hypertrophied myocardium is now understood to be accountable from a severe reduction in the oxidation of endogenous TAG, which cannot be recruited by adrenergic challenge.6 Thus, TAG as an energy source becomes essentially unavailable, coinciding with the reduced long chain fatty acid incorporation into the TAG pool. In the present study, restoration of TAG content, however, was not linked to oxidation of the endogenous, unlabeled fatty acid. Indeed, activation of pyruvate oxidation with DCA outpaced the oxidation of the endogenous long chain fatty acids in the normal, sham-operated hearts. Similarly, the additional oxidation of pyruvate with DCA treatment in hypertrophied hearts would out compete any potential contributions from endogenous fatty acids. Therefore, although the present protocol elucidated a mechanistic link between increased anaplerosis and limitations in TAG synthesis in hypertrophied hearts, the reason for the loss of TAG as a source of fatty acid oxidation remains unclear.

However, this study does clarify the different fate of glycolytic end products for oxidative metabolism in the hypertrophied versus normal cardiomyocyte. We show here that malic enzyme content is increased in hypertrophied hearts. We have also confirmed our previous findings that neither pyruvate carboxylase nor propionyl-CoA carboxylase content were changed with hypertrophy (Figure 3C), further supporting the role of malic enzyme for catalyzing the increase anaplerotic flux. However, as may be expected by the expression of both pyruvate carboxylase and propionyl-CoA carboxylase, some baseline anaplerotic flux can be expected through both of these enzyme pathways.

Our previous study identified the source of this anaplerosis as glycolytic.5 This finding is important, because previous reports indicate that the oxidation of glycolytic end products does not keep pace with increased glycolytic activity in the hypertrophied heart.11,29 We have since indicated that a significant portion of glycolytic end product, not observed to enter the citric acid cycle via PDH, actually does enter the citric acid cycle via anaplerosis.5 This alternate pathway can be easily overlooked because of measurements based on the release and capture of radiolabeled CO2 that is not sensitive to anaplerotic flux. Pyruvate carboxylation via malic enzyme not only bypasses CO2 release at PDH, but also consumes a CO2 group during carboxylation. The specific sensitivity of 13C NMR detection schemes to anaplerotic activity enabled elucidation these events, linking malic enzyme activity to limitations in TAG content in hypertrophied hearts.

Yet, the potential for some mismatch between glycolysis and glucose oxidation does exist, as supported by the observation of increased shuttling of cytosolic NADH in response to pressure overload.4 Other laboratories have used DCA to augment glucose oxidation in hypertrophied hearts, but activating PDH did not induce control levels of “coupling” between glycolysis and the oxidation of pyruvate via PDH.10,20 However, without benefit of measuring anaplerosis or NADH shuttling into mitochondria, at the time the authors could only conclude that “other mechanisms are responsible.”4,5,10

In summary, this study elucidates the role of glycolytic pyruvate, produced from glucose, as the carbon source for increased anaplerotic flux in the hypertrophied heart. Tissue contents of both enzyme and product implicate malic enzyme as the catalyst of this anaplerotic reaction. In addition to the reduced efficiency in reducing equivalent production from pyruvate conversion to malate, compared to acetyl-CoA conversion to citrate, additional evidence is now provided that links malic enzyme-dependent anaplerosis to reduced TAG content in hypertrophied myocardium. Thus, with the normalization of dP/dt in hypertrophied hearts with metabolic intervention, these findings provide evidence that changes in intermediary metabolism during cardiac hypertrophy because of altered metabolic enzyme expression are maladaptive, and are detrimental to contractility. However, the role of such maladaptive enzyme expression in the transition from compensatory to decompensatory hypertrophy remains to be fully established.